MvaT调控铜绿假单胞菌吩嗪的合成机制

Regulation of MvaT on phenazine synthesis in Pseudomonas aeruginosa

【背景】属于H-NS家族的MvaT转录因子参与了铜绿假单胞菌的许多重要代谢过程,如吩嗪合成代谢,但其调控方式仍不十分明确。【目的】确定转录调控因子MvaT是否直接调控铜绿假单胞菌的吩嗪合成过程,即该蛋白是否可以直接结合2个吩嗪-1-羧酸合成基因簇(phzA1G1和phzA2G2)与3个分支转化基因(phzH、phzS和phzM)的上游启动子区域。【方法】以铜绿假单胞菌SJTD-1和其mvaT基因敲除突变株SJTD-1(ΔmvaT)为研究对象,检测其在不同培养基条件下吩嗪化合物的合成量差异。通过体外异源表达与亲和纯化,获得重组蛋白MvaT。利用凝胶阻滞实验,确定MvaT重组蛋白对5个吩嗪代谢基因簇/基因上游启动子的结合情况。【结果】mvaT基因敲除突变株SJTD-1(ΔmvaT)的吩嗪产量较野生型显著提升。MvaT重组蛋白被有效表达与纯化,体外凝胶阻滞实验结果显示,该重组蛋白可与phzA1G1、phzA2G2、phzM、phzS和phzH的上游启动子区域均发生特异性结合。其中,重组蛋白MvaT与phzA1G1和phzA2G2的结合区域位于其上游启动子的200 bp以内,而该蛋白与phzM、phzS和phzH的结合区域则位于其上游启动子的100 bp以内。【结论】MvaT蛋白通过直接结合吩嗪合成代谢基因的上游启动子区域来直接调控假单胞菌的吩嗪类化合物合成。

[Background] MvaT protein belonging to the H-NS transcription factor family is involved in many important metabolic processes of Pseudomonas aeruginosa,such as phenazine synthesis pathway.However,up to now its regulation mode is still unclear.[Objective] The goal of this work was to determine if MvaT could directly regulate the two phenazine-1-carboxylic acid synthesis gene clusters(phzA1 G1 and phzA2 G2) and three transforming genes(phzH,phzS and phzM) of phenazine compounds,by detecting the binding capability of MvaT to the promoter regions of these genes.[Methods]Pseudomonas aeruginosa SJTD-1 was used as target and the mvaT-knockout strain SJTD-1(ΔmvaT) was constructed by homologous recombination.The yield of phenazine compounds of the two strains in different media was detected.Further the recombinant MvaT protein was obtained by the heterologous expression and affinity purification.The electrophoretic mobility shift assay(EMSA) was performed to determine if the recombinant MvaT protein could bind to the promoter regions of the five phenazine synthetic gene clusters/genes.[Results] The yield of phenazine compounds of strain SJTD-1(ΔmvaT) was significantly higher than that of the wild type SJTD-1 strain.The recombinant MvaT protein could be expressed and purified efficiently.In vitro EMSA results indicated that the recombinant MvaT protein could directly bind to the promoter regions of phenazine synthetic gene clusters/genes.The binding sites of MvaT to the promoter of phzA1 G1 and phzA2 G2 gene clusters were within the upstream 200 bp region,and that to the promoter of phzM,phzS and phzH genes were within its upstream 100 bp region.[Conclusion] MvaT protein can directly bind the upstream promoter regions of the five phenazine synthetic gene clusters/genes and regulate the synthesis of phenazine compounds.